It is widely known that a central nervous system (CNS)-neuron in higher organisms such as human is not capable of regeneration once injured. Therefore, one who has received an injury on his (her) spinal cord due to, for example, a traffic accident, is compelled to spend the rest of his (her) life in a hemiplegic state. On the contrary, it is known that a peripheral nervous system (PNS)-neuron retains a vigorous regeneration ability even in those higher organisms, and therefore, neurons in a limb, when disconnected, can gradually regenerate with a concomitant recovery of their function.
In the early nineteen-eighties, a group of Aguayo et al. found that when PNS-neuron is experimentally grafted into an injured CNS-neuron in a higher organism, axon growth of CNS-neuron is induced. This observation demonstrated that CNS-neuron in higher organisms which had been generally considered not to have a regeneration ability can regenerate if a suitable environment is provided (Nature, 284, 264-265 (1980), Science, 214, 931-933 (1981)). That report suggests a possibility that in CNS of higher organisms, there may exist a factor, namable “CNS-neuron regeneration inhibitor”, which inhibits the regeneration of CNS-neuron, and that a release from such inhibition may allow the regeneration of CNS-neurons. This suggestion paved the way for a CNS-neuron regeneration therapy.
In 1988; a group of Schwab et al. demonstrated that there existed such CNS-neuron regeneration inhibitor among proteins derived from CNS myelin. They also succeeded in purifying, though partially, a protein having said CNS-neuron regeneration inhibition activity, and named this protein fraction NI35/250 (Annu. Rev. Neurosci., 16, 565-595 (1993)), although no one has succeeded in its isolation, identification and gene cloning yet. In addition, they immunized animals with the partially purified NI35/250, and succeeded in obtaining an antibody (IN-1) having a neutralizing activity. This antibody is capable of recognizing the band for NI35/250 in Western blotting, and capable of staining, in an immunostaining, the region to which NI35/250 is supposed to be distributed. Furthermore, they demonstrated that administration of this antibody to an animal experimentally received an injury on its spinal cord has promoted regeneration of axons in spinal cord, though partially, within 2-3 weeks, and restored its function within 2-3 months (Nature, 343, 269-272 (1990), Nature, 378, 498-501 (1995)). These findings are of great value, because they experimentally demonstrated that there existed a CNS-neuron regeneration inhibitor as suggested by Aguayo et al. (supra) and that CNS-neuron can be regenerated by inhibiting the activity of said inhibitor. The above antibody is, however, directed not to human but to rat NI35/250, and exhibits a low stability and specificity. In addition, although regeneration of CNS-neuron was observed as described above by administering said antibody, its effect was so partial and incomplete that not all of the motor functions could be restored. It is, therefore, believed essential in solving these problems to identify the gene for NI35/250 or similar CNS-neuron regeneration inhibitor, and, based on knowledges of molecular biology, neuroscience and the like, develop an antagonist more effectively inhibiting the CNS-neuron regeneration inhibition activity, or develop a method for inhibiting the expression of the gene for said regeneration inhibitor.
Apart from the above, the nervous system, whether it is central or peripheral, requires formation of a complicated neural network among neurons or between neurons and peripheral receivers or effectors during development, that is, in the stage of embryo or fetus, in order to precisely carry out its principal functions, i.e., to transfer and process the information. To establish the neural network, an ingenious mechanism is necessary, which precisely guides a growing neurite to the target site locating remote therefrom.
It has been hitherto believed that a factor which positively controls the neurite outgrowth, such as neurite growth promoter and neurite growth attractant may play a major role in the formation of the neural network. However, it is now being demonstrated by recent studies on the mechanism of the network formation that the opposite factor, that is, a negative factor having an outgrowth inhibition activity is important for an accurate guidance (Cell, 78, 353-356 (1994)).
A representative factor having such an outgrowth inhibition activity is a protein called “Semaphorin”. Semaphorin firstly discovered is Fasciclin IV found in grasshopper. Collapsin (latterly named Collapsin I) was subsequently discovered in chick (Cell, 75, 217-227 (1993); Neuron, 9, 831-845 (1992)). To date, more than 10 genes belonging to the Semaphorin family have been reported in a wide range of species covering insects such as drosophila and beetle, human, and viruses (Cell, 81, 471-474 (1996)). These Semaphorins characteristically contain in their amino acid sequences similar structures called semaphorin domains each consisting of about 600 amino acids (Neuron, 14, 941-948 (1995); Cell, 75, 1389-1399 (1993)). However, the homologies of the primary amino acid sequences in semaphorin domains among these Semaphorin genes are 80-20%, and not necessarily high.
Of these Semaphorins, functions have been verified for only a few, including, for example, Fasciclin IV of grasshopper, Semaphorins I and II of drosophila, Collapsin of chick, and Semaphorin III which corresponds to Collapsin in mammals. All of these Semaphorins are known to inhibit neurite outgrowth or synapsis formation. In particular, Semaphorin III has been reported to have an activity collapsing in a short time the growth cone of cultured neuron (growth-cone collapse activity) in vitro (Neuron, 14, 941-948 (1996); Neuron, 14, 949-959 (1995); Cell, 81, 631-639 (1995); Cell, 75% 1389-1399 (1993); Cell, 75, 217-227 (1993); Neuron, 9, 831-845 (1992)).
Although it is now being demonstrated, as described above, that known Semaphorins have a growth-cone collapse activity and a neurite outgrowth inhibition activity during development, and play a role in giving an accurate guidance to neuron, it is not evident at present whether or not the Semaphorins exert some function not only during development but also in the adult, and less evident whether or not Semaphorins play a role as a CNS-neuron regeneration inhibitor. Of course, since known Semaphorins have been shown to be a negative to guidance factor inhibiting neurite outgrowth, it would not be unreasonable to consider said Semaphorins as a candidate for a CNS-neuron regeneration inhibitor (Nature, 378, 439-440 (1995)). However, it has been shown by in vitro experiments that Semaphorin III (Sema III), only one Semaphorin of higher organisms of which function has been analyzed, exerts its neurite-outgrowth inhibition activity on a sensory neuron and sympathetic neuron both of which are peripheral, but not on a retinal neuron which is central (Cell, 75, 217-227 (1993)). In addition, Northern analysis on the distribution of Sema III expression in the adult carried out by the present inventors has revealed that it is expressed mainly in peripheral tissues (see Reference example 2 below). It is therefore hardly believed that Sema III having such features has a function as a CNS-neuron regeneration inhibitor.
Problem to be Solved by the Invention
The present invention aims to provide Semaphorin W, a novel Semaphorin belonging to the Semaphorin family, and a gene therefor, and to provide pharmaceutical agents for neural diseases, in particular for regeneration of CNS-neuron, and related diagnostic agents or laboratory reagents. More specifically, the present invention aims to provide Semaphorin W which inhibits neurite outgrowth and a gene therefor, as well as other Semaphorins hybridizing to said Semaphorin W gene, modified proteins or partial peptides of said Semaphorin W, antibodies against said Semaphorin W, antisense nucleotides against said Semaphorin W gene, and use of such substances as pharmaceutical or diagnostic agents or laboratory reagents. The present invention further aims to provide a method of screening for Semaphorin W antagonists employing said Semaphorin W, Semaphorin W antagonists obtained by said screening method, pharmaceutical agents comprising such antagonists, and transgenic animals regarding said Semaphorin W.
Means of Solving the Problem
In order to provide pharmaceutical agents for neural diseases, in particular for regeneration of CNS-neuron, and related diagnostic agents or laboratory reagents, the present inventors have planed to identify a novel Semaphorin which has not yet been cloned. In particular, the present inventors have paid their attention to the similarity between the in vitro activities of the above-described NI35/250 and the negative guidance factor Semaphorin, i.e., to the fact that NI35/250 has a growth-cone collapse activity and a neurite-growth inhibition activity in vitro (J. Neurosci., 8, 2381-2393 (1988); Science, 259, 80 (1993)), while known Semaphorins similarly possess a neurite-growth inhibition activity, and particularly Semaphorin III has also a growth-cone collapse activity. This suggested to the inventors the possibility that unknown Semaphorins which have not yet been identified may include the one inhibiting regeneration of CNS-neuron. Specifically, the present inventors' idea was that Semaphorin having those characteristics that 1) it is highly expressed in the CNS of the adult, but 2) it is poorly expressed in other tissues where regeneration of neuron (or neurite outgrowth) is not inhibited, such as peripheral tissues in the adult and fetus tissues, has not been identified yet, and if one can identify a new unknown Semaphorin having such characteristics, the Semaphorin might be involved in inhibition of regeneration of CNS-neuron.
First of all, the inventors have searched EST (Expressed Sequence Tags) database for DNA sequences encoding the amino acids relatively well conserved among previously reported Semaphorin genes. As a consequence, the DNA fragment T09073 (SEQ ID NO:15) was identified, which encodes, as a partial sequence, a sequence: Gln-Asp-Pro-Val0Cys-Ala-Trp (SEQ ID NO:12), similar to that consisting of seven amino acids extremely well conserved among Semaphorins (Gln(or Arg)-Asp-Pro-Tyr-Cys-Ala(or Gly)-Trp) (SEQ ID NO:13).
The T09073 gave a sequence information as to only 364 bp and contained undetermined bases, and further, the open reading frame could not be determined. It was, therefore, utterly impossible at that stage to conclude that T09073 corresponds to part of a gene encoding “Semaphorin”. Furthermore, although T09073 has been submitted to the database as a sequence derived from a human child brain cDNA library, it was unknown whether or not the sequence is expressed in the fetus or peripheral tissues of the adult, and therefore, it could not be concluded that the sequence corresponds to part of a novel Semaphorin gene specifically expressed in the CNS.
Thus, the present inventors firstly carried out Northern analysis using a DNA fragment consisting of 196 base pairs from the 5′ region of T09073 as a probe, in order to check the distribution of expression of a gene containing T09073. As a result, it was found that the gene corresponding to T09073 was highly and widely expressed in CNS tissues in the adult, whereas among the other tissues, it was expressed only in the lung and spleen of the adult throughout the fetal and postnatal periods. It was thus demonstrated that the gene exhibited a distribution pattern of expression expected for a novel Semaphorin gene at which the present inventors aimed.
Next, the present inventors cloned the gene containing T09073 in full length in order to ascertain whether it is a novel Semaphorin or not. Specifically, a rat cDNA library was screened using the above DNA fragment consisting of 196 base pairs as a probe. As a result, the gene thus cloned proved to be a novel Semaphorin gene having a sequence characteristic to Semaphorins. We named this novel Semaphorin “Semaphorin W”.
Further analysis revealed that Semaphorin W has an effect as a novel Semaphorin gene at which the present inventors aimed, that is, an inhibition activity against neurite outgrowth, especially an inhibition activity for CNS-neurons.
Semaphorin W of the present invention appears to be involved in inhibition of regeneration of CNS-neuron in the adult, since it is highly expressed in CNS in the adult and has an inhibitory effect on CNS-neurons as described above. Semaphorin W may be used to screen for Semaphorin W antagonists, and those antagonists identified in such screening system are expected to promote regeneration of CNS-neuron. Similarly, antisense DNAs or RNAs against Semaphorin W gene are also expected to promote regeneration of CNS-neuron as well as the above antagonists.
In addition, in view of the fact that Semaphorin W of the present invention has also an inhibitory effect on PNS-neurons, it may be used as a therapeutic or diagnostic agent for pains or immune diseases such as atopic dermatitis, by administering it to peripheral tissues, which results in the inhibition of neurite outgrowth of PNS-neuron. Furthermore, Semaphorin W is a novel Semaphorin belonging to the Semaphorin family which has unprecedented features regarding its distribution of expression and its effect as described above. Semaphorin W, therefore, serves as an important research material or a laboratory reagent.
The present invention has been completed on the basis of the above findings.
Thus, the gist of the present invention relates to:    (1) a gene encoding the following protein (a) or (b):            (a) Semaphorin W protein comprising the amino acid sequence shown in SEQ ID NO: 3,        (b) a protein which comprises an amino acid sequence wherein one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 3, and which protein inhibits neurite outgrowth;            (2) a gene comprising the following DNA (c), (d), or (e):            (c) Semaphorin W DNA comprising the base sequence shown in SEQ ID NO: 1 or 2,        (d) DNA which hybridizes under stringent conditions to DNA comprising the base sequence shown in SEQ ID NO: 1 or 2, and which encodes a protein inhibiting neurite outgrowth,        (e) DNA of the above item (d) which contains the base sequence shown in SEQ ID NO: 4 or 5 and/or the base sequence shown in SEQ ID NO: 10;            (3) a gene comprising DNA which hybridizes under stringent conditions to DNA comprising the base sequence shown in SEQ ID NO: 7, and which encodes a protein having a semaphorin domain;    (4) a protein obtained by expressing a gene of any one of the above items (1) to (3);    (5) a gene encoding a protein comprising an amino acid sequence wherein one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 3, and which protein promotes neurite outgrowth;    (6) a protein obtained by expressing a gene of the above item (5);    (7) DNA which is cloned from a human cDNA library or a human genomic library, and which hybridizes under stringent conditions to DNA comprising at least part of DNA consisting of the base sequence shown in SEQ ID NO: 1, 4, or 10;    (8) an expression plasmid which expresses either a gene of any one of the above items (1) to (3) and (6), or DNA of the above item (7);    (9) a transformant transformed with an expression plasmid of the above item (8);    (10) a process for producing a recombinant protein, which process comprises culturing a transformant of the above item (9), and recovering the recombinant protein expressed;    (11) a peptide comprising a segment of at least six or more amino acids of a protein of the above item (4) or (6);    (12) a peptide of the above item (11) which promotes neurite outgrowth;    (13) a peptide of the above item (11) characterized in that it contains the glutamic acid residue at position 204 of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid residue corresponding to the position of said glutamic acid residue;    (14) an antisense nucleotide, or chemically modified variant thereof, which is directed against a segment of at least eight or more bases of a gene of any one of the above items (1) to (3), or of DNA of the above item (7);    (15) an antisense nucleotide, or chemically modified variant thereof, of the above item (14), characterized in that it inhibits expression of a protein of the above item (4);    (16) an antibody against a protein of the above item (4) or (6), or against a peptide of any one of the above items (11) to (13);    (17) a pharmaceutical agent comprising, as an active ingredient, a gene of any one of the above items (1) to (3) and (5), DNA of the above item (7), a protein of the above item (4) or (6), a peptide of any one of the above items (11) to (13), an antisense nucleotide or chemically modified variant thereof of the above item (14) or (16), or an antibody of the above item (16);    (18) a method of screening for Semaphorin W antagonists, characterized in that it employs a protein of the above item (4);    (19) Semaphorin W antagonist obtained by the screening method of the above item (18);    (20) Semaphorin W antagonist of the above item (19) which comprises a protein of the above item (6), a peptide of any one of the above items (11) to (13), or an antibody of the above item (16);    (21) a CNS-neuron regeneration promoter, characterized in that it contains at least one of the antisense nucleotides or chemically modified variants thereof of the above item (14) or (15), or Semaphorin W antagonists of the above item (19) or (20);    (22) a neurite outgrowth inhibitor for PNS-neuron, characterized in that it contains at least one of the proteins of the above item (4); and    (23) a transgenic animal in which either a gene of any one of the above items (1) to (3) and (5), or DNA of the above item (7) has been artificially inserted into its chromosome, or has been knocked out.